Description of Research Expertise

My laboratory studies the cell-intrinsic, molecular mechanisms that T lymphocytes use to decode extracellular signals and translate these into the appropriate immune vs. tolerant responses. Our research has focused primarily on 1) the regulation of T cell activation by factors coupled to the receptors for antigen, B7 and IL-2, which provide the co-signals necessary for T cell activation and anergy avoidance, and 2) the molecular basis for failed cytokine gene transcription in regulatory T cells and T cells rendered anergic by activation in the absence of the appropriate costimulatory signals. We have defined a series of intracellular proteins expressed in quiescent T cells that sense signals from costimulatory and growth factor receptors.
One of these proteins is p27kip1, a potent inhibitor of cyclin-dependent kinases (CDK) that is highly expressed in resting cells. This cell cycle regulator is targeted for degradation in CD4+ T cells receiving signals through CD28 and IL-2R, and is elevated when T cells receive anergizing signals through the TCR alone. We showed in a series of studies that p27kip1 is required for the induction of anergy in vitro and the establishment of acquired immunological tolerance in vivo, as T cells genetically deficient for the cdkn1b gene that encodes p27kip1 are resistant to anergy induction in vitro, and costimulatory blockade in vivo fails to induce tolerance to fully-mismatched cardiac allografts in mice that lack p27kip1. Using CDK2-deficient mice and shRNA approaches, our most recent work has established that p27kip1 promotes anergy in large part through opposing the activity of CDK2, a kinase that licenses CD4+ T cells that receive costimulatory signals to avoid anergy. CDK2-deficient T cells exhibit a phenotype resembling anergy even after receiving full costimulation through the TCR and CD28, and mice lacking CDK2 are more susceptible to transplantation tolerance. We are currently screening for downstream targets of CDK2 in CD4+ T cells to determine how CDK2 couples TCR/CD28 signals to the il2 gene.
We have identified another protein (and potential CDK2 substrate) that senses costimulatory signals in resting T cells as Ikaros, a zinc finger transcriptional repressor previously known to be involved in lymphocyte development. In a series of four papers in the past four years, we have shown that anergy in both CD4+ and CD8+ T cells is an epigenetically imprinted state that is reinforced by active histone deacetylation and DNA methylation at cytokine genes. More recently, we have shown that Ikaros is in large part responsible for targeting these processes to the il2 and ifnγ genes and for silencing of the cytokines in tolerant T cells. Ikaros is bound directly to the il2 promoter in mature, naïve CD4+ T cells, where it recruits co-repressor complexes containing histone deacetylases (HDAC) and DNA methyltransferases (DNMT). Ikaros is required to maintain the il2 promoter in an epigenetically quiescent state, is required for repression of il2 transcription in the absence of CD28, and is required for the induction of anergy in conventional CD4+ T cells. Interestingly, in work submitted for publication, we also show that Ikaros directly silences the gene encoding T-bet in developing Th2 cells, and loss of Ikaros function results in failed silencing of the il2 and ifnγ genes in this T helper lineage. Together, these studies suggest a general role for Ikaros in cytokine gene silencing under a variety of circumstances where the production of pro-inflammatory cytokines is not appropriate. We are currently investigating the network of genes regulated by Ikaros in mature T cells, and how Ikaros activity is controlled by extracellular signals.
My laboratory has also been very interested in Foxp3, a lineage-specific protein required for the development, suppressive function, and anergic phenotype of regulatory T cells (Treg). When we began studying this factor in 4-5 years ago, it was not clear how Foxp3 established the Treg transcriptional program. For instance, evidence at the time suggested that Foxp3 could only repress gene expression, and it was not clear whether the ‘winged-helix’ domain of Foxp3 could bind directly to DNA. This left unanswered the major question of how Foxp3 activates Treg-specific genes, which constitutes a larger subset than the repressed genes. We began to address this question using a chromatin immunoprecipitation approach to study binding of ectopically expressed, tagged Foxp3 (antibodies against Foxp3 were not available at the time) to the endogenous regulatory regions of genes whose expression was known to be Foxp3-responsive. We showed that Foxp3 binds directly to the cytokines genes that are repressed in Treg (e.g., il2 and ifnγ). Interestingly, our studies demonstrated that Foxp3 is not bound constitutively to these cytokine genes, but requires active signaling from the antigen receptor and can be inhibited by cyclosporine A. This indicates that Foxp3 is not constitutively active, but responds to extracellular signals to repress transcription. These results were confirmed by the Rao group, who showed that Foxp3 binds cooperatively to the il2 gene with the cyclosporine-sensitive transcription factor NFAT1. We also showed that Foxp3 binding to these cytokine genes is accompanied by strong histone de-acetylation that could be blocked by HDAC inhibition, indicating that Foxp3 actively silences cytokine genes by recruiting co-repressor complexes. Importantly, we also confirmed that Foxp3 binds directly to the promoters of several Foxp3-induced genes, such as cd25, ctla4 and gitr, and instead of inducing histone de-acetylation in these regions, Foxp3 binding leads to histone hyper-acetylation. These studies made it clear that both Foxp3-repressed and Foxp3-induced genes are direct Foxp3 targets (a finding confirmed by subsequent genome-scale studies by the Rudensky and Von Boehmer & Young groups), and that Foxp3 can function as both a transcriptional co-repressor, and as a transcriptional co-activator. In recent studies that we are submitting for publication, we found that Foxp3 and Ikaros physically interact and cooperate to repress cytokine genes in regulatory T cells. These data show that Ikaros is required for anergy in both conventional and regulatory T cells, and suggest that Ikaros partnering may form a biochemical switch that determines whether Foxp3 represses vs. induces a given target gene.

The decision between anergy and productive T cell activation is made by a series of intracellular proteins that actively oppose induction of the il2 gene, but are inactivated by signals from CD28 and/or the IL-2 receptor. Ikaros and p27kip1 fulfill this role, as these factors are present in naïve T cells (i.e., their expression is not anergy-specific), and are required for the induction of anergy. In the absence of Ikaros, quiescent T cells are able to produce IL-2 and differentiate in response to signals from the TCR alone. T cells lacking p27kip1 still require CD28 costimulation for IL-2 production during the primary stimulus, but during restimulation behave as though they received an anergy avoidance signal. Ikaros is also functions downstream of p27kip1 and CDK2 to decode signals from the IL-2R, as T cells with reduced Ikaros activity cannot be anergized by IL-2/IL-2R blockade. Therefore, I refer to these factors as anergy avoidance sensors, and together with costimulatory and mitogenic signals, they form a ‘lock and key’ mechanism that ensures that only T cells meeting their antigen under the appropriate inflammatory conditions gain immune effector function. Foxp3 in regulatory T cells is a specialized anergy factor that also acts as a sensor of mitogenic signals. However, instead of being inactivated by anergy avoidance signals (like p27kip1 and Ikaros), Foxp3 responds to antigenic signaling by binding to and actively silencing pro-inflammatory cytokine genes in conjunction with NFAT1 and Ikaros. The action of these (and other) factors is why antigenic stimulation in the absence of costimulation is not a neutral event leading to ignorance, but rather results in the loss of function characteristic of anergy. Our current work and immediate future directions include identifying TCR/CD28/IL-2R-coupled kinases or enzymes that regulate Ikaros transcriptional function, the significance of Foxp3-Ikaros partnering on Treg function, the nature of the co-repressor and co-activator complexes recruited by Foxp3 or Foxp3-Ikaros complexes to Treg-specific genes, what functions of Foxp3 are affected by IPEX mutations, whether these factors function downstream of CDK2 in T cells, and how these events impact immunity vs. tolerance in autoimmune and transplantation models.